WO2023155291A1 - 一种唾液和鼻腔双样本一体化加样的检测技术及其应用 - Google Patents

一种唾液和鼻腔双样本一体化加样的检测技术及其应用 Download PDF

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WO2023155291A1
WO2023155291A1 PCT/CN2022/087800 CN2022087800W WO2023155291A1 WO 2023155291 A1 WO2023155291 A1 WO 2023155291A1 CN 2022087800 W CN2022087800 W CN 2022087800W WO 2023155291 A1 WO2023155291 A1 WO 2023155291A1
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detection
sampling
saliva
sample
nasal cavity
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PCT/CN2022/087800
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English (en)
French (fr)
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刘默文
刘杰
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嘉兴康源科泰科技发展有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B10/0045—Devices for taking samples of body liquids
    • A61B10/0051—Devices for taking samples of body liquids for taking saliva or sputum samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the invention relates to the technical field of medical devices, in particular to a detection technology for simultaneous sampling of saliva and nasal cavity double samples and integrated sample addition detection and its application.
  • Immunological detection technology is an experimental method designed to determine antigens, antibodies, immune cells and chemical components based on the principle of applied immunology. , pharmaceutical analysis, food and industrial analysis samples. Commonly used detection techniques include immunoturbidity technology, solid-phase enzyme immunochromatography assay technology, chemiluminescence detection technology, immunofluorescence labeling technology, quantum dot immunoassay technology, colloidal gold immunoassay technology, latex microsphere immunoassay technology, dot immunoassay detection technology, etc. High sensitivity, rapidity, convenience, miniaturization, full quantification, and automation are the current development trends of clinical immunoassay technology.
  • Point-of-care testing is currently the fastest-growing branch, and chromatography immunoassay is the most commonly used detection method, and colloidal gold, fluorescent lateral flow chromatography immunoassay and latex microsphere immunoassay technology are the most widely used , but the methods used are generally single-sample sampling and detection methods.
  • nasal swab or anterior nasal swab is the main sampling method for most respiratory infectious diseases at present.
  • the extract is used for pretreatment of the sample.
  • Saliva is also one of the test samples, but its viral load is lower than that of nasal swabs, which affects the positive detection rate of cases.
  • the purpose of the present invention is to provide a kind of integrated detection technology and its application of simultaneously sampling double samples of saliva and nasal cavity and adding samples for detection. Check quality.
  • the present invention provides a detection technology for integrated sampling of saliva and nasal cavity double samples, comprising the following steps: using a saliva sampling structure to collect a saliva sample into a saliva collection container, using a nasal cavity sampling structure to extend the sampling head into the The nasal cavity sample is collected in the nasal cavity, the sampling head with the nasal cavity sample collected is directly placed into the collected saliva sample, stirred and eluted to form a mixed liquid phase of the saliva sample and the nasal cavity sample, and the mixed liquid phase is soaked and absorbed to the sampling head of the nasal cavity sampling structure, and then directly insert the sampling head into the detection sample collection structure arranged on the chromatography detection structure, and the mixed liquid phase is directly loaded into the On the sample pad of the detection test strip of the liquid flow traffic, flow through the marker binding pad, the solid phase chromatography detection membrane and the water absorption pad, by observing the amount of the marker that is indirectly captured on the solid phase chromatography detection membrane, read Test results, complete the testing process.
  • the saliva sampling structure is a container with an opening.
  • one end of the sampling head inserted into the nasal cavity is fixedly provided with a water-absorbing layer.
  • the tomographic detection structure is a shell-shaped structure, and the tomographic detection structure is provided with a sampling head insertion port toward the direction of the sampling head, and the detection sample collection structure is located on the layer Inside the analysis detection structure, a liquid flow channel is formed between the detection sample collection structure and the detection test strip, wherein the detection test strip is arranged in sequence from the sampling head insertion port to the distal end with the sample pads and marks A substance-binding pad, a solid-phase chromatography detection membrane and a water-absorbent pad, and an observation window for viewing the detection results of the detection test strip is opened above the chromatography detection structure.
  • the saliva sampling structure is composed of a funnel-shaped saliva collection structure with a large top and a small bottom and a test tube-shaped saliva collection container, which can be disassembled.
  • the nasal cavity sampling structure includes a sampling handle, and the sampling head is directly fixedly connected to the sampling handle.
  • the solid-phase chromatography detection membrane is at least one of nitrocellulose membrane, polyvinylidene fluoride membrane, nylon membrane, and DEAE cellulose membrane.
  • the marker in the marker-binding pad is at least one of colloidal metal, fluorescent marker, and latex microspheres.
  • the water-absorbent layer of the sampling head is made of water-absorbent and water-insoluble materials, including natural and modified polymer superabsorbent resins and artificially synthesized water-absorbent resins, starch series, cellulose series, other natural product series, Polyvinyl salt series, polyvinyl alcohol series and polyoxyethylene series, etc.
  • Some of the polyvinyl alcohol series products have the characteristics of drying and hardening, and water absorption and softening, such as flocking, sponge, polyester fiber, polyester, cotton, and more man-made fibers etc.
  • the detection technique includes a detection-aided liquid phase, which is an aqueous buffered saline solution with no denaturing agent.
  • the detection test strip includes a sample pad, a marker binding pad, a filter membrane pad, a nitrocellulose membrane pad and a water-absorbing pad that are sequentially pasted on the PVC negative film, and the detection test strip is placed in a chromatographic detection structure.
  • the present invention is a detection technology that adopts double sample sampling of saliva and nasal cavity, and collects double samples of saliva and nasal cavity at the same time during detection, which can significantly improve the positive detection rate of cases and improve the detection sensitivity of diseases, unlike the existing The market can only improve the sensitivity of the detection reagent itself but cannot change the pathogen load of the sample to be tested.
  • nasal swab or anterior nasal swab is the main sampling method for most respiratory infectious diseases at present.
  • the extract is used for pretreatment of the sample.
  • Saliva is one of the test samples, but its viral load is lower than that of nasal swabs, which affects the positive detection rate of cases.
  • the present invention adopts double-sample sampling of saliva and nasal cavity, which can not only superimpose the viral load in the two collected samples, but also make saliva into an effective solvent by optimizing the detection system, thereby realizing the need for no extraction
  • the rapid detection of liquid realizes the detection accuracy, convenience, rapidity and simple operation.
  • the nasal cavity sampling structure of the present invention is not only a collection structure for the nasal cavity to be detected, but also a structure for adding samples when detecting saliva and nasal cavity to be detected.
  • the substance is the solvent liquid phase of the substance to be tested in the nasal cavity and the mobile liquid phase for the detection of the substance to be tested, and a creative multifunctional design is carried out for the sampling structure.
  • the saliva sampling structure of the present invention is a funnel-shaped structure with a collection tube, so that the saliva can be spit out through the funnel and directly enter the collection tube, which is easy to operate and avoids pollution.
  • the nasal cavity sampling structure of the present invention includes a sampling handle and a sampling head, wherein the sampling head has dual functions of sampling and detection and loading, and realizes various advantages such as convenient collection, sample transfer and sample loading safety.
  • the sampling head containing saliva collection and nasal cavity collection is directly inserted into the detection sample collection structure of the chromatography detection structure, and the mixed liquid phase absorbed by soaking is directly loaded into the detection sample collection structure.
  • the detection reaction is started on the sample pad of the detection test strip with liquid flow communication, which not only ensures the accuracy of sampling, but also ensures that the structure and detection function of the detection test strip are not affected during the sampling process.
  • the present invention uses detection test strips as detection components, and is applicable to various detection technologies with lateral flow as the main technology, such as colloidal gold, fluorescent immunoassay, latex microsphere immunoassay, etc., expanding the scope of application.
  • the present invention is equipped with an auxiliary detection liquid phase, which can be used for samples with insufficient liquid phase, and improves the practicality of detection.
  • the invention has simple operation steps and is easy to realize home use or self-testing. It is not only convenient to use and reduces the waste of raw materials, but also significantly improves work efficiency, and can be applied to many fields of professional and amateur testing.
  • Fig. 1 is the schematic flow chart of detection technology of the present invention
  • Fig. 2 is the schematic diagram of nasal cavity sampling structure of the present invention.
  • Fig. 3 is the structural representation of saliva sampling of the present invention.
  • Fig. 4 is a schematic diagram of the chromatographic detection structure of the present invention.
  • Fig. 5 is a schematic diagram of the detection sample collection structure of the present invention.
  • the detection technical process of the present invention includes three modules: a saliva sampling structure, a nasal cavity sampling structure, and a chromatography detection structure.
  • the sampling structure extends the sampling head into the nasal cavity to collect nasal samples, directly puts the sampling head with nasal samples into the collected saliva samples, stirs and elutes, and forms a mixed liquid phase of saliva samples and nasal samples, and makes the mixed liquid
  • the liquid phase is soaked and absorbed into the sampling head of the nasal cavity sampling structure, and then the sampling head is directly inserted into the detection sample collection structure set in the chromatography detection structure, and the mixed liquid phase is directly loaded into the detection test paper that has fluid communication with the detection sample collection structure
  • On the sample pad of the strip it flows through the marker-binding pad, the solid-phase chromatography detection membrane and the water-absorbing pad.
  • the nasal cavity sampling structure of the present invention includes a sampling handle 1, a sampling head fixing structure 2, and a sampling head 3, wherein the sampling head 3 is fixed on the sampling handle 1 through the sampling head fixing structure 2, and the sampling handle 1 is held by hand during operation , the sampling head 3 is placed in the anterior nasal cavity for sampling.
  • the funnel-shaped saliva collection structure 4 is selected as the saliva sampling structure of the present invention, and the small opening of the funnel of the saliva collection structure 4 forms a detachable connection with the saliva collection tube 5, and the saliva is spit to the saliva collection structure 4 through the mouth during use. , and then flow into the saliva collection tube 5, and then insert the sampling head 3 carrying the nasal cavity sample into the saliva collection tube 5 after sampling, agitate the saliva against the wall, form a mixture of nasal cavity sample and saliva sample, soak and absorb it into the sampling head 3, complete the sampling process of double-sample integration.
  • the chromatography detection structure of the present invention includes a detection connection structure 6, an observation window 8, a detection housing cover 9, a detection housing base 10, a detection connection sampling head insertion port 11, and a detection sample collection structure 12 and detection test strip 13.
  • the detection test strip 13 is prepared by the colloidal gold method, and the colloidal gold binding of the sample pad and the colloidal gold marker coated with the colloidal gold marker are pasted successively on the PVC negative film.
  • the detection test strip 13 is prepared by the fluorescent immunoassay method, and samples are sequentially pasted on the PVC film Pads, fluorescent microsphere binding pads coated with fluorescent markers, nitrocellulose membrane pads and absorbent pads coated with non-labeled capture reagents; when the detection technology of the present invention is a latex microsphere immunoassay structure, the detection test strip 13 Prepared by latex microsphere immunization method, on the PVC film, the sample pad, the latex microsphere binding pad coated with latex microsphere markers, the nitrocellulose membrane pad coated with non-labeled capture reagent, and the water-absorbing pad are pasted in sequence .
  • the saliva sampling structure When in use, take out the saliva sampling structure, nasal cavity sampling structure and chromatography detection structure, use the saliva sampling structure to collect the saliva sample through the saliva collection structure 4, and collect it into the saliva collection tube 5; hold the sampling handle 1, and extend the sampling head 3 into the nasal cavity
  • To collect a nasal cavity sample directly put the sampling head 3 with the nasal cavity sample into the collected saliva sample, stir and elute to form a mixed liquid phase of the saliva sample and nasal cavity sample, and soak and absorb the mixed liquid phase into the sampling head 3
  • the sampling head 3 is directly inserted into the detection sample collection structure 12 of the chromatographic detection structure through the detection connection sampling head insertion port 11, and the mixed liquid phase is directly loaded to the detection sample collection structure 12 that has liquid flow communication.
  • On the sample pad of the test strip 13 flow through the marker binding pad, the solid phase chromatography detection membrane and the water absorption pad, observe the situation that the marker on the solid phase chromatography detection membrane is indirectly captured, read the detection result, and complete the detection process .
  • Experiment 1 Rapid detection experiment of the new coronavirus antigen by the immunocolloidal gold method of the present invention:
  • Adopt conventional immune colloidal gold detection technology double-antibody sandwich method to prepare detection test strips use the detection technology of the present invention to prepare detection test strips for new coronavirus antigen detection experiments, wherein the colloidal gold marker on the detection line T of the detection test strips indicates that the antibody is 10ug/ml anti-new coronavirus N protein monoclonal antibody, using colloidal gold particles with a particle size of 50nm, coated on the glass cellulose membrane colloidal gold binding pad; the capture antibody of the detection line T of the test strip is 1.0mg/ml The paired anti-new coronavirus N protein monoclonal antibody is coated on the nitrocellulose membrane pad; the capture antibody of the quality control line C of the test strip is 1.0 mg/ml goat anti-mouse IgG polyclonal antibody, coated on the On the nitrocellulose membrane pad, it is used to capture the colloidal gold-labeled anti-new coronavirus N protein monoclonal antibody that has not been specifically captured. Paste absorbent pads and colloidal gold marker binding pads
  • Adopt Solidworks to design the upper cover of the detection shell, the base of the detection shell, the nasal cavity sampling structure and the saliva sampling structure of the present invention's chromatography detection structure, and use 3D to print the samples, paste the sponge sampling head on the sampling head fixed structure, and adopt the preservation tube used in small experiments As a saliva collection tube, the prepared chromatographic detection structure samples were used for experimental detection.
  • Method 1 Take 150ul solution 1), add it into a test tube, take a nasal cavity sampling structure, collect nasal cavity samples from healthy people, insert the sampling head into the solution 1), stir and absorb the solution 1), and then pass through Insert the insertion port into the detection buckle, let it stand for 20 minutes, check the observation window, and read the color development result on the test strip.
  • Method 2 Take 100ul of saliva from a healthy person without N protein, add it to the test tube, take the nasal cavity sampling structure, collect a healthy human nasal cavity sample, and then take 50ul of the solution 2) Add it to the sampling head after nasal cavity sampling, insert the sampling head into In the saliva of the blank healthy person, stir and absorb the saliva solution of the blank healthy person, then insert it into the detection buckle through the insertion port, let it stand for 20 minutes, check the observation window, and read the color development result on the test strip.
  • the quality control line C of the test strip has color, and the test line T has no color, which is negative; the quality control line C has color, and the test line T also has color, which is positive.
  • the final concentration of antigen N protein solution is 1.0, 0.1, 0.05, 0.01ng/ml
  • the detection results of both methods are positive, and when the concentration is 0.001ng/ml or below, the detection results are all negative.
  • the conventional latex microsphere immunochromatography technology double-antibody sandwich method was used to prepare the detection test strip, and the chromatography detection structure of the present invention was used to prepare the detection kit for the new coronavirus antigen detection experiment.
  • the latex microsphere label indicator antibody of the detection line T of the test strip is a 50ug/ml anti-new coronavirus N protein monoclonal antibody, which is coated on the glass cellulose membrane binding pad; the capture antibody of the detection line T of the test strip A paired anti-new coronavirus N protein monoclonal antibody of 1.0mg/ml, coated on a nitrocellulose membrane pad; the capture antibody of the quality control line C of the test strip is 1.0mg/ml goat anti-mouse IgG polyclonal Antibodies, coated on nitrocellulose membrane pads, are used to capture latex microspheres that have not been specifically captured to mark anti-new coronavirus N protein monoclonal antibodies.
  • Method 1 Take 150ul solution 1), add it into a test tube, take a nasal cavity sampling structure, collect nasal cavity samples from healthy people, insert the sampling head into the solution 1), stir and absorb the solution 1), and then pass through Insert the insertion port into the detection buckle, let it stand for 20 minutes, check the observation window, and read the color development result on the test strip.
  • Method 2 Take 100ul of saliva from a healthy person without N protein, add it to the test tube, take the nasal cavity sampling structure, collect a healthy human nasal cavity sample, and then take 50ul of the solution 2) Add it to the sampling head after nasal cavity sampling, insert the sampling head into In the saliva of the blank healthy person, stir and absorb the saliva solution of the blank healthy person, then insert it into the detection buckle through the insertion port, let it stand for 20 minutes, check the observation window, and read the color development result on the test strip.
  • the quality control line C of the test strip has color, and the test line T has no color, which is negative; the quality control line C has color, and the test line T also has color, which is positive.
  • concentration of antigen N protein solution is 1.0, 0.1, 0.05, 0.01ng/ml
  • the detection results of the two methods are all positive, and when the concentration is 0.001ng/ml or below, the detection results are all negative.
  • Test materials for control Nucleic acid detection by RT-qPCR.
  • the product uses BGI New Coronavirus Nucleic Acid Real-Time Fluorescent RT-PCR Kit (Product No. MFG030010), which was granted FDA Emergency Use Authorization (EUA) in March 2020, and the packaging size is 50 pieces Reaction/kit, the manufacturer is Huada Genomics, and the detection equipment is ABI 7500 fluorescent quantitative PCR detector, MGISP-960 high-throughput automatic extraction and liquid handling workstation and MGI Easy nucleic acid extraction kit (Cat. No. 1000020261).
  • EUA Emergency Use Authorization
  • the performance of the novel coronavirus antigen chromatographic detection structure prepared by the detection technology of the present invention is evaluated, and compared with the detection results of RT-qPCR.
  • the samples used for RT-qPCR detection shall be sampled by nasal swabs according to the instructions, and shall be stored in non-inactivated UTM preservation solution; Invented nasal cavity sampling structure sampling, put into non-inactivated UTM preservation solution for preservation; samples used for saliva antigen chromatography detection structure detection, adopt saliva sampling structure of the present invention, directly spit saliva into and collect into saliva collection tube;
  • the nasal cavity sampling structure of the present invention is used to collect nasal cavity samples, the saliva sampling structure of the present invention is used to directly spit saliva into the saliva collection tube, and then the nasal cavity sampling structure is directly put into the collected saliva, Stir to mix and soak and absorb the saliva onto the sampling head for detection.
  • Table 3 the clinical comparative study of double-sample sampling antigen detection and PCR nucleic acid detection of the present invention
  • Tables 1, 2, and 3 are the detection results of 85 subjects.
  • the positive coincidence rate of the sample collected by the nasal cavity sampling structure of the present invention is 91.2%, and the negative coincidence rate is 91.2%. 100%;
  • the positive coincidence rate of the sample collected by the saliva sampling structure of the present invention is 70.3% with the antigen chromatography detection structure detection, and the negative coincidence rate is 100%;
  • adopt the double-sample detection technology of the present invention that is, directly spit saliva into and collect into the saliva collection tube, then put the sampling head with the nasal cavity sample directly into the saliva collection tube, stir and mix, soak and absorb the saliva onto the sampling head, then insert the sampling head directly into the antigen chromatography detection structure, start the detection, and observe
  • the positive coincidence rate of detection was 100%, and the negative coincidence rate was 100%.
  • the statistical analysis showed a significant difference, p ⁇ 0.01. It shows that the detection technology of the present invention can significantly improve the accuracy of antigen chromatography

Abstract

一种唾液和鼻腔双样本一体化加样的检测方法，通过唾液采样结构（4）采集唾液样本，鼻腔采样结构采集鼻腔内样本，将采集有鼻腔样本的鼻腔采样结构采样头放入唾液样本内混合，制成唾液样本与鼻腔样本的混合液并吸收到鼻腔采样结构的采样头（3）上，然后将采样头（3）直接插入层析检测结构（7）的检测样本采集结构（12）内，直接加载混合液至检测试纸条（13）的样本垫上，启动并完成检测过程。该方案用于胶体金、荧光免疫、乳胶微球等多种快速免疫检测产品的开发，提高了免疫检测产品的检测效率、便捷性和准确性，具有重要的临床意义。

Description

一种唾液和鼻腔双样本一体化加样的检测技术及其应用 技术领域
本发明涉及医疗器械技术领域，尤其涉及一种唾液和鼻腔双样本同时采样并进行一体化加样检测的检测技术及其应用。
背景技术
免疫学检测技术是根据应用免疫学原理设计的测定抗原、抗体、免疫细胞及化学成分等的实验手段，广泛用于检测来源于人体和动物体可进行疾病诊断和健康检测的样品以及用于环境、药物分析、食品和工业分析的样品。常用的检测技术有免疫浊度技术、固相酶免疫层析测定技术、化学发光检测技术、免疫荧光标记技术、量子点免疫检测技术、胶体金免疫检测技术、乳胶微球免疫检测技术、斑点免疫检测技术等。高灵敏度、快速、便捷、小型化、全定量、自动化是目前临床免疫检测技术的发展趋势。床旁检测(POCT)是目前发展最快的一个分支，层析免疫检测是其最常用的检测方法，胶体金、荧光侧向流层析免疫检测和乳胶微球免疫检测技术的产品使用最广，但所采用的方法一般均为单一样本的采样检测方法。众所周知，鼻拭子或前鼻拭子检测是目前多数呼吸道传染性疾病的主要采样方式，呼吸系统的病毒载量高，但缺陷是采集和使用的可接受性差和便捷性低，检测过程均需要提取液对样本进行前处理。唾液也是检测样本之一，但其病毒载量相较鼻拭子而言比较低，影响病例的阳性检出率。因此，开发唾液和鼻腔双样本采样并检测的技术，不仅可以做到两种采集样本中病毒载量的叠加，同时通过对检测系统优化处理，将唾液做成了一种有效的溶剂，进而实现了不需要提取液的快速检测，实现了检测的准确性、便捷性、快速性和操作简单性，将有助于临床检测产品的普及使用，提高诊疗防治质量，具有重要的应用价值。
发明内容
本发明的目的是提供一种唾液和鼻腔双样本同时采样并加样检测的一体化检测技术及其应用，与现有技术比较，具有检测灵敏度高、便捷、快速、防止污染等的特点，提高检测质量。
针对上述目的，本发明提供的一种唾液和鼻腔双样本一体化加样的检测技术，包括 如下步骤：采用唾液采样结构将唾液样本采集至唾液采集容器内，采用鼻腔采样结构将采样头伸入鼻腔内采集鼻腔样本，直接将采集有鼻腔样本的所述采样头置入至采集的唾液样本内，搅拌洗脱，形成唾液样本与鼻腔样本的混合液相，并使所述混合液相浸泡吸收到所述鼻腔采样结构的采样头上，然后将所述采样头直接插入设置于层析检测结构上的检测样本采集结构内，所述混合液相被直接加载至与所述检测样本采集结构具有液流交通的检测试纸条的样本垫上，流经标记物结合垫、固相层析检测膜和吸水垫，通过观察所述固相层析检测膜上标记物被间接捕获的量，读取检测结果，完成检测过程。
上述所述检测技术中，所述唾液采样结构为设有开口的容器。
上述所述检测技术中，所述采样头插入鼻腔的一端固定设置有吸水层。
上述所述检测技术中，所述层析检测结构为一种壳状结构，所述层析检测结构朝向所述采样头的方向开设有采样头插入口，所述检测样本采集结构位于所述层析检测结构内部，所述检测样本采集结构与所述检测试纸条之间形成液流通道，其中所述检测试纸条从所述采样头插入口向远端依次排列所述样本垫、标记物结合垫、固相层析检测膜和吸水垫，所述层析检测结构上方开设有查看所述检测试纸条检测结果的观察窗。
上述所述检测技术中，所述唾液采样结构由漏斗状的上大下小的唾液采集结构和试管状的唾液收集容器组成，两者间可拆装。
上述所述检测技术中，所述鼻腔采样结构包括采样手柄，所述采样头与所述采样手柄直接固定连接。
上述所述检测技术中，所述固相层析检测膜为硝酸纤维素膜、聚偏氟乙烯膜、尼龙膜、DEAE纤维素膜中的至少一种。
上述所述检测技术中，所述标记物结合垫中的标记物为胶体金属、荧光标记物、乳胶微球中的至少一种。
本发明所述检测技术在免疫检测产品的研究开发中的应用。
所述采样头的吸水层由具有吸水性且不溶于水的材料制成，包括天然及改性高分 子高吸水性树脂和人工合成吸水性树脂，淀粉系列、纤维素系列、其他天然产物系列、聚乙烯酸盐系列、聚乙烯醇系列和聚氧乙烯系列等，其中部分聚乙烯醇系列产品拥有干燥变硬、吸水变软的特性，如植绒、海绵、聚酯纤维、涤纶、棉花、多种人造纤维等。
所述检测技术包含有助检液相，所述助检液相为非变性剂的水质缓冲盐溶液。
所述检测试纸条包括在PVC底片上依次粘贴的样本垫、标记物结合垫、过滤膜垫、硝酸纤维素膜垫和吸水垫，所述检测试纸条放置于层析检测结构内。
本发明由于采取以上技术方案，其具有以下优点：
1、本发明为采用唾液和鼻腔双样本采样的检测技术，检测时同时采集唾液和鼻腔的双重样本，这样可以显著提高病例的阳性检出率，提高疾病的检测灵敏度，而并不像现有市场只能提高检测试剂本身的灵敏度而无法改变待检样本的病原体载量。众所周知，鼻拭子或前鼻拭子检测是目前多数呼吸道传染性疾病的主要采样方式，呼吸系统的病毒载量高，但缺陷是采集和使用的可接受性低和便捷性差，检测过程均需要提取液对样本进行前处理。唾液作为检测样本之一，但其病毒载量相较鼻拭子而言比较低，影响病例的阳性检出率。本发明采用唾液和鼻腔双样本采样，不仅可以做到两种采集样本中病毒载量的叠加，同时通过对检测系统优化处理，将唾液做成了一种有效的溶剂，进而实现了不需要提取液的快速检测，实现了检测准确性、便捷性、快速性和操作简单性。
2、本发明鼻腔采样结构既是作为鼻腔待检物的采集结构，同时又是对唾液和鼻腔待检物进行检测时加样的结构，唾液采样结构既是唾液待检物的采集结构，同时唾液采集物又是鼻腔待检物的溶媒液相以及对待检物进行检测的流动液相，对采样结构进行了创造性的多功能设计。
3、本发明唾液采样结构为带有收集管的漏斗状结构，使唾液经漏斗吐入后直接进入收集管，操作简便，避免污染。
4、本发明的鼻腔采样结构包括采样手柄和采样头，其中采样头具有采样和检测上样的双重功能，实现了采集便捷、样本转移和上样安全等多种优势。
5、本发明将含有唾液采集物和鼻腔采集物的采样头直接插入设置于层析检测结 构的检测样本采集结构内，被浸泡吸收的所述混合液相直接加载至与所述检测样本采集结构具有液流交通的检测试纸条的样本垫上，启动检测反应，不仅保证了取样的准确性，而且保证了在取样过程中不影响检测试纸条的结构及检测功能。
6、本发明采用检测试纸条作为检测部件，适用于多种以侧向流为主要技术的检测技术，如胶体金、荧光免疫、乳胶微球免疫检测等，拓展了应用范围。
7、本发明配备了助检液相，可用于液相不充分的样本，提高了检测的实用性。
8、本发明操作步骤简单，易于实现家庭化使用或自我检测，不仅使用方便、减少原料的浪费，同时也显著提高工作效率，可以应用于专业和业余检测的诸多领域。
附图说明
图1为本发明的检测技术流程示意图；
图2为本发明的鼻腔采样结构示意图；
图3为本发明的唾液采样结构示意图；
图4为本发明的层析检测结构示意图；
图5为本发明的检测样本采集结构示意图。
图中标记如下：
采样手柄1；采样头固定结构2；采样头3；唾液采集结构4；唾液收集管5；检测连接结构6；层析检测结构7；观察窗8；检测外壳上盖9；检测外壳底座10，检测连接采样头插入口11；检测样本采集结构12；检测试纸条13。
具体实施方式
为更进一步阐述本发明为达成预定目的所采取的技术手段及功效，以下所述实施例结合附图对本发明进行进一步说明，但本发明并不局限于以下说明。
如图1所示，本发明的检测技术流程，包括唾液采样结构、鼻腔采样结构和层析检测结构3个模块，操作步骤包括：采用唾液采样结构将唾液样本采集至唾液采集容器内，采用鼻腔采样结构将采样头伸入鼻腔内采集鼻腔样本，直接将采集有鼻腔样本 的采样头置入至采集的唾液样本内，搅拌洗脱，形成唾液样本与鼻腔样本的混合液相，并使混合液相浸泡吸收到鼻腔采样结构的采样头上，然后将采样头直接插入设置于层析检测结构的检测样本采集结构内，混合液相直接加载至与检测样本采集结构具有液流交通的检测试纸条的样本垫上，流经标记物结合垫、固相层析检测膜和吸水垫，通过观察固相层析检测膜上标记物被间接捕获的情况，读取检测结果，完成检测过程。
如图2所示，本发明鼻腔采样结构，包括采样手柄1、采样头固定结构2、采样头3、其中采样头3经采样头固定结构2固定于采样手柄1上，操作时手持采样手柄1，将采样头3置于前鼻腔进行采样。
如图3所示，本发明唾液采样结构选用漏斗状的唾液采集结构4，唾液采集结构4的漏斗小口与唾液收集管5形成可拆装式连接，使用时唾液经口腔吐至唾液采集结构4，之后流入唾液收集管5内，然后将完成采样后的载有鼻腔样本的采样头3插入至唾液收集管5内，贴壁搅动唾液，形成鼻腔样本与唾液样本的混合物并浸泡吸收至采样头3上，完成双样本一体化的采样过程。
如图4、图5所示，本发明的层析检测结构，包括检测连接结构6、观察窗8、检测外壳上盖9、检测外壳底座10、检测连接采样头插入口11、检测样本采集结构12和检测试纸条13。
在实际操作时，当本发明检测技术为胶体金免疫检测结构时，检测试纸条13用胶体金法制备，在PVC底片上依次粘贴有样本垫、包被有胶体金标记物的胶体金结合垫、包被有非标记捕获试剂的硝酸纤维素膜垫和吸水垫；当本发明检测技术为荧光免疫检测结构时，检测试纸条13用荧光免疫法制备，在PVC底片上依次粘贴有样本垫、包被有荧光标记物的荧光微球结合垫、包被有非标记捕获试剂的硝酸纤维素膜垫和吸水垫；当本发明检测技术为乳胶微球免疫检测结构时，检测试纸条13用乳胶微球免疫法制备，在PVC底片上依次粘贴有样本垫、包被有乳胶微球标记物的乳胶微球结合垫、包被有非标记捕获试剂的硝酸纤维素膜垫和吸水垫。使用时，取出唾液采样结构、鼻腔采样结构和层析检测结构，采用唾液采样结构将唾液样本经唾液采集结构4，采集入唾液收集管5内；手持采样手柄1，将采样头3伸入鼻腔内采集鼻腔样本，直接将采集有鼻腔样本的采样头3置入至采集的唾液样本内，搅拌洗脱，形成唾液样本与鼻腔样本的混合液相，并使混合液相浸泡吸收到采样头3上，然后将采样头3经检测连接采样头插入口11直接插入设置于层析检测结构的检测样本采集结构12内，混合液相直接被加载至与检测样本采集结构12具有液流交通的检测试纸条13的样本垫上， 流经标记物结合垫、固相层析检测膜和吸水垫，通过观察固相层析检测膜上标记物被间接捕获的情况，读取检测结果，完成检测过程。
本发明的实验研究：下述实验说明本发明的检测技术及其效果，但不是对本发明的限定。下述实验中所使用的实验方法如无特殊说明，均为常规方法。所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实验一：本发明免疫胶体金法新冠病毒抗原快速检测实验：
一、检测试纸条制备：
采用常规免疫胶体金检测技术双抗体夹心法制备检测试纸条，采用本发明检测技术制备检测试纸条进行新冠病毒抗原检测实验，其中检测试纸条的检测线T的胶体金标记指示抗体为10ug/ml的抗新冠病毒N蛋白单克隆抗体，采用粒径50nm的胶体金颗粒，包被在玻璃纤维素膜胶体金结合垫上；检测试纸条的检测线T的捕获抗体为1.0mg/ml的配对的抗新冠病毒N蛋白单克隆抗体，包被在硝酸纤维素膜垫上；检测试纸条的质控线C的捕获抗体为1.0mg/ml的羊抗鼠IgG多克隆抗体，包被在硝酸纤维素膜垫上，用于捕获未被特异性捕获的胶体金标记抗新冠病毒N蛋白单克隆抗体。在硝酸纤维素膜垫印膜两端分别粘贴吸水垫及胶体金标记结合垫，在胶体金标记结合垫一侧粘贴样本垫。置粘贴好的检测片于切条机上，切成3.5mm的检测试纸条。
二、本发明层析检测结构制备：
采用Solidworks设计本发明层析检测结构的检测外壳上盖、检测外壳底座，鼻腔采样结构和唾液采样结构，并用3D打印样品，在采样头固定结构上粘贴海绵采样头，采用小型实验使用的保存管作为唾液收集管，制备的层析检测结构样品用于实验检测。
三、实验方法及结果：
实验时取上述制备的检测试纸条和层析检测结构，装配成检测扣卡，将装配好的检测扣卡放入有干燥剂的铝箔密封袋内，在封口机上封口，加贴标签。配制两种待检溶液，1)用健康人唾液配置不同浓度的重组新冠病毒抗原N蛋白溶液；2)用非灭活型病毒保存液(UTM)配置3倍于溶液1)浓度的不同浓度的重组新冠病毒抗原N蛋白溶液。分别采用两种方法进行检测，方法一：取150ul溶液1)，加入试管内，取鼻腔采样结构，采集健康人鼻腔样本，将采样头插入至溶液1)内，搅动吸收溶液1)，然后经插入口插入检测扣卡内，静置20分钟，查看观察窗，读取检测试纸条上的显 色结果。方法二：取100ul不加N蛋白的健康人唾液，加入试管内，取鼻腔采样结构，采集健康人鼻腔样本，再取50ul溶液2)加至鼻腔采样后的采样头上，将采样头插入至空白健康人唾液内，搅动吸收空白健康人唾液溶液，然后经插入口插入检测扣卡内，静置20分钟，查看观察窗，读取检测试纸条上的显色结果。检测试纸条质控线C有显色，检测线T无显色，为阴性；质控线C有显色，检测线T也有显色，为阳性。抗原N蛋白溶液终浓度为1.0、0.1、0.05、0.01ng/ml时，两种方法的检测结果均为阳性，浓度为0.001ng/ml及以下时，检测结果则均为阴性。
实验二：本发明乳胶微球免疫层析法新冠病毒抗原快速检测实验：
一、检测试纸条制备：
采用常规乳胶微球免疫层析技术双抗体夹心法制备检测试纸条，采用本发明层析检测结构制备检测试剂盒进行新冠病毒抗原检测实验，其中乳胶微球均采用度生物300nm红色微球，检测试纸条的检测线T的乳胶微球标记指示抗体为50ug/ml的抗新冠病毒N蛋白单克隆抗体，包被在玻璃纤维素膜结合垫上；检测试纸条的检测线T的捕获抗体为1.0mg/ml的配对的抗新冠病毒N蛋白单克隆抗体，包被在硝酸纤维素膜垫上；检测试纸条的质控线C的捕获抗体为1.0mg/ml的羊抗鼠IgG多克隆抗体，包被在硝酸纤维素膜垫上，用于捕获未被特异性捕获的乳胶微球标记抗新冠病毒N蛋白单克隆抗体。在硝酸纤维素膜垫印膜两端分别粘贴吸水垫及乳胶微球标记结合垫，在乳胶微球标记结合垫一侧粘贴样本垫。置粘贴好的检测片于切条机上，切成3.5mm的检测试纸条。
二、本发明层析检测结构制备：
同“实验一”制备。
三、实验方法及结果：
实验时取上述制备的检测试纸条和层析检测结构，装配成检测扣卡，将装配好的检测扣卡放入有干燥剂的铝箔密封袋内，在封口机上封口，加贴标签。同上，配制两种待检溶液，1)用健康人唾液配置不同浓度的重组新冠病毒抗原N蛋白溶液；2)用非灭活型病毒保存液(UTM)配置3倍于溶液1)浓度的不同浓度的重组新冠病毒抗原N蛋白溶液。分别采用两种方法进行检测，方法一：取150ul溶液1)，加入试管内，取鼻腔采样结构，采集健康人鼻腔样本，将采样头插入至溶液1)内，搅动吸收溶液1)，然后经插入口插入检测扣卡内，静置20分钟，查看观察窗，读取检测试 纸条上的显色结果。方法二：取100ul不加N蛋白的健康人唾液，加入试管内，取鼻腔采样结构，采集健康人鼻腔样本，再取50ul溶液2)加至鼻腔采样后的采样头上，将采样头插入至空白健康人唾液内，搅动吸收空白健康人唾液溶液，然后经插入口插入检测扣卡内，静置20分钟，查看观察窗，读取检测试纸条上的显色结果。检测试纸条质控线C有显色，检测线T无显色，为阴性；质控线C有显色，检测线T也有显色，为阳性。抗原N蛋白溶液浓度为1.0、0.1、0.05、0.01ng/ml时，两种方法的检测结果均为阳性，浓度为0.001ng/ml及以下时，检测结果则均为阴性。
实验三：本发明单样本和双样本一体化采样对新冠病毒临床检测性能的影响
一、检测试纸条制备：
同“实验二”制备。
二、本发明检测技术层析检测结构制备：
同“实验一”制备。
三、对照用核酸检测试剂：
对照用检测材料：通过RT-qPCR进行核酸检测，产品采用BGI新冠病毒核酸实时荧光RT-PCR试剂盒(货号MFG030010)，于2020年3月获得FDA紧急使用授权(EUA)，包装规格为50个反应/试剂盒，制造商为华大基因，检测仪器为ABI 7500荧光定量PCR检测仪、MGISP-960高通量自动提取和液体处理工作站和MGI Easy核酸提取试剂盒(货号1000020261)。
四、实验方法及结果：
对采用本发明检测技术制备的新冠病毒抗原层析检测结构的性能进行评估，并与RT-qPCR的检测结果进行比较。
1、样本选择：
选择新冠病毒感染疑似病例和感染接触人群，根据RT-qPCR核酸检测结果，将样本分为阳性组和阴性组。同时通过RT-qPCR和抗原层析检测结构检测鼻拭子样本、唾液样本以及本发明唾液与鼻拭子采集物的混合样本，并将两种结果进行分析比较。
2、样品收集和处理
用于RT-qPCR检测的样本，采用前鼻拭子采样方式，按照说明书要求采样后，放入非灭活UTM保存液内保存；用于鼻拭子抗原层析检测结构检测的样本，采用本发明鼻腔采样结构采样，放入非灭活UTM保存液内保存；用于唾液抗原层析检测结构检测 的样本，采用本发明唾液采样结构，直接将唾液吐入并收集至唾液收集管内；用于本发明双样本检测的样本，采用本发明鼻腔采样结构采集鼻腔样本，采用本发明唾液采样结构直接将唾液吐入并收集至唾液收集管内，然后将鼻腔采样结构直接放入采集好的唾液内，搅拌混合并将唾液浸泡吸收至采样头上，备用检测。
五、结果及分析
对85例受试者进行比对检测，其中37例为临床新冠病毒感染人群样本，48例为健康人群样本。检测结果如表1至表3。
表1、鼻拭子采样抗原检测与PCR核酸检测的临床比对研究
Figure PCTCN2022087800-appb-000001
表2、唾液采样抗原检测与PCR核酸检测的临床比对研究
Figure PCTCN2022087800-appb-000002
表3、本发明双样本采样抗原检测与PCR核酸检测的临床比对研究
Figure PCTCN2022087800-appb-000003
表1、2、3为85例受试者的检测结果，与RT-qPCR检测结果比较，本发明鼻腔 采样结构采集的样本用抗原层析检测结构检测的阳性符合率为91.2％，阴性符合率为100％；本发明唾液采样结构采集的样本用抗原层析检测结构检测的阳性符合率为70.3％，阴性符合率为100％；采用本发明双样本检测技术，即直接将唾液吐入并收集至唾液收集管内，然后将采集有鼻腔样本的采样头直接放入唾液收集管内，搅拌混合后，唾液浸泡吸收至采样头上，然后将采样头直接插入抗原层析检测结构中，启动检测，观察结果，检测的阳性符合率为100％，阴性符合率为100％。与两种单独采样检测技术比较，经统计学分析差异显著，p＜0.01。表明本发明的检测技术可以显著提高抗原层析检测结构检测的准确性，同时还不影响检测特异性。

Claims (9)

  1. 一种唾液和鼻腔双样本一体化加样的检测技术，包括如下步骤：采用唾液采样结构将唾液样本采集至唾液采集容器内，采用鼻腔采样结构将采样头伸入鼻腔内采集鼻腔样本，直接将采集有鼻腔样本的所述采样头置入至采集的唾液样本内，搅拌洗脱，形成唾液样本与鼻腔样本的混合液相，并使所述混合液相浸泡吸收到所述鼻腔采样结构的采样头上，然后将所述采样头直接插入设置于层析检测结构上的检测样本采集结构内，所述混合液相被直接加载至与所述检测样本采集结构具有液流交通的检测试纸条的样本垫上，流经标记物结合垫、固相层析检测膜和吸水垫，通过观察所述固相层析检测膜上标记物被间接捕获的量，读取检测结果，完成检测过程。
  2. 根据权利要求1所述检测技术，其特征在于：所述唾液采样结构为设有开口的容器。
  3. 根据权利要求1所述检测技术，其特征在于：所述采样头插入鼻腔的一端固定设置有吸水层。
  4. 根据权利要求1所述检测技术，其特征在于：所述层析检测结构为一种壳状结构，所述层析检测结构朝向所述采样头的方向开设有采样头插入口，所述检测样本采集结构位于所述层析检测结构内部，所述检测样本采集结构与所述检测试纸条之间形成液流通道，其中所述检测试纸条从所述采样头插入口向远端依次排列所述样本垫、标记物结合垫、固相层析检测膜和吸水垫，所述层析检测结构上方开设有查看所述检测试纸条检测结果的观察窗。
  5. 根据权利要求1所述检测技术，其特征在于：所述唾液采样结构由漏斗状的上大下小的唾液采集结构和试管状的唾液收集容器组成，两者间可拆装。
  6. 根据权利要求1所述检测技术，其特征在于：所述鼻腔采样结构包括采样手柄，所述采样头与所述采样手柄直接连接。
  7. 根据权利要求1所述检测技术，其特征在于：所述固相层析检测膜为硝酸纤维素膜、聚偏氟乙烯膜、尼龙膜、DEAE纤维素膜中的至少一种。
  8. 根据权利要求1所述检测技术，其特征在于：所述标记物结合垫中的标记物为胶体金属、荧光标记物、乳胶微球中的至少一种。
  9. 权利要求1所述检测技术在免疫检测产品的研究开发中的应用。
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